In liquid chromatography, a compound is broken down into its components in a chromatographic column, so that these components can be analyzed or further processed.
For the analysis of complex compounds, e.g., peptides or proteins, there is frequently the problem that the separating power is not sufficient to be able to distinguish or selectively analyze the individual components. Therefore, for such compounds, several separating steps are used one after the other. Here, it is especially efficient to use different material properties for the individual separations. In this way, the compound can first be broken down into substance groups, which can then be analyzed individually in more detail. Such methods are known as so-called two-dimensional, three-dimensional, or multi-dimensional chromatography methods (2D or 3D chromatography).
Here, one distinguishes between online methods, in which the separating steps follow one another directly in time, as well as offline techniques, in which the separating steps are performed independently of each other in time. The latter has the advantage that the individual separating steps can be optimized individually and independently of each other.
However, offline techniques require higher expense because the components (fractions), already separated in the preceding steps, must be buffered in separate form in order to be able to feed them to the following separating processes at the given time. The process is designated as fractioning. The collection of the fractions is usually performed with a separate device, the fraction collector. This divides the fractions into a corresponding number of fraction collecting or holding containers.
Usually, one would like to collect a certain, preset liquid volume in each fraction-collecting container.
Automated analysis systems usually have available automatic sample injectors, which hold a plurality of samples to be analyzed and which can feed these in series according to the analysis system. Such a sample injector is described, for example, in U.S. Pat. No. 5,814,742. Because such sample injectors form the basis of both the invention and the state of the art, the components are explained with reference to the simplified, schematic representation in FIG. 1.
Through the input capillary 1, the liquid flow delivered by a pump is led into the sample injector and leaves it via the output capillary 5. Here, a chromatographic separating column is connected, which is designated below as column 20. This is usually located outside of the sample injector. In the liquid flow there is a 6-port switch valve 2, which has available two switch positions. The position shown in FIG. 1 is designated below as “position a-b,” where the port a is connected to b, c to d, and e to f. The second position is designated as “position f-a” and connects port b to c, d to e, and f to a. Thus, the liquid flow from the input capillary 1 can directly pass the switch valve b. The sample containers 7 contain the samples to be analyzed, which can be removed via a sample needle 6. A mechanism not shown in FIG. 1 allows the sample needle 6 and the sample container 7 to move relative to each other, so that the sample needles 6 can approach any sample container 7 and can be inserted into this container in order to remove the appropriate sample.
In the shown position a-b of the switch valve 2, the following components are connected in series: a dosing syringe 4, a sample loop 3, a connection capillary 8, and also the sample needle 6. While the sample needle 6 is inserted into a sample container 7, sample material can be removed from the sample container 7 through suction with the dosing syringe 4 and can be drawn, in particular, into the sample loop 3. By switching the switch valve 2 to position f-a, the sample loop 3 between the input capillary 1 and the output capillary 5 is switched and the sample material is transported with the liquid flow in the direction towards the column 20. Adding the sample into the liquid flow is designated as injection. In this described way, the samples to be analyzed can be injected in any sequence. During the injection, the dosing syringe 4 is now connected directly to the sample needle 6. In order to empty the dosing syringe again, the sample needle 6 can be moved to a waste port 9, which receives the excess solution and feeds it to a container for waste.
Furthermore, automatic sample injectors usually contain devices in order to clean the sample needle 6 from any adhering residue of the stored sample. These are not shown in FIG. 1 for the sake of clarity. In this way, diverting sample material from one sample container 7 to the next should be avoided.
The injection principle described above has the disadvantage that not only the sample loop 3, but also at least the sample needle 6 and the capillary 8 must be filled with sample material. In this way, considerably more sample material is needed than is required for the injection. Another disadvantage is that the sample amount to be injected is fixed by the size of the sample loop. Indeed, it would be possible, in principle, to draw the syringe 4 so far that the sample loop 3 is only partially filled with sample material, but this requires, e.g., exact knowledge of the volume of the capillary 8 and the sample needle 6.
These two disadvantages can be avoided through the so-called “micro-liter pickup” method, which is also described in U.S. Pat. No. 5,814,742. Here, one of the sample containers 7 contains a transport liquid, i.e., solution without sample material. Before suctioning the sample, a certain amount of this transport liquid is suctioned from this container. Then the sample needle 6 is moved into the sample container 7, which contains the desired sample. Now only the desired, small amount of sample is suctioned, wherein, in each case, its volume must be smaller than the interior volume of the sample loop 3. Then the sample needle is again moved into the container with transport liquid and this is sufficiently suctioned so that the “sample plug” enclosed between transport liquid is located in the sample loop 3. In this method, no sample material is lost and small sample volumes can also be injected, which are significantly less than the interior volume of the sample loop.
In principle, a sample injector can also be used for sample fractioning, since the sample injector already has available a holding device for sample containers and a corresponding positioning ability for the sample needle. Thus, the necessary components for distributing the fractions to a number of fraction-collecting containers are already present. Corresponding solutions have been published, for example, in:
LC Packings, Baarsjaweg 154, 1057 HM Amsterdam, Netherlands: Application Note FAMOS μ-Sampling Workstation, No. 10, Appendix I: “2-D capillary LC separations,” and
Gilson Inc., Middleton, Wis. 53562-0027, USA: Product Brochure 233 XL System for On-Line Column Switching, pg. 3: “Fraction collection in sealed vials . . . .”
These known solutions differ from each other through device-specific features, for example, in construction; however, all require an additional switch valve for switching between the sample injecting and fraction collecting functions. Because such switch valves are very expensive due to the strict requirements, this means considerable additional expense.
Such a solution is shown schematically in FIG. 2 for better understanding of the present invention:
The first separating step is performed in column 20, i.e., the collecting fractions are sequentially discharged at the output of the column 20. These fractions can now be distributed to a number of fraction-collecting containers.
For this purpose, the sample injector as shown in FIG. 2 must be expanded. Here the connection capillary 8 is separated into two connection capillaries 801 and 802 and a second switch valve 13 is inserted in-between. The fractions discharged from the column 20 are led back into the sample injector via the return capillary 10 and can be fed to the fraction-collecting containers 12 via the second switch valve 13, the connection capillary 801, and the sample needle 6. The waste capillary 11 leads to a waste container, in which the solution coming before or after the arrival of the fractions, as well as undesired fraction components, can be collected. According to the position of the second switch valve 13, the arrangement works either as a sample injector or as a fraction collector.
The operation is as follows: first both switch valves 2 and 13 are in position a-b, as shown in FIG. 2. A sample is removed from the sample container in the way as described above and injected by switching the switch valve 2. It is led into the column 20 via the outlet capillary 5, where the first separating step is performed. Passing through the capillaries and the separation itself take a certain amount of time, so that the separated fractions reach the sample injector at a later time via the return capillary 10. Now the sample needle 6 is positioned on the desired fraction-collecting container 12 and the second switch valve 13 is switched to position f-a, so that the return capillary 10 is connected to the sample needle 6 via the connection capillary 8. The fraction is stored in the corresponding fraction-collecting container 12. For the next fraction, the sample needle 6 is positioned on the next fraction-collecting container 12.